Rubisco: Active-site characterization and mechanistic implications

Title: Rubisco: Active-site characterization and mechanistic implications

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Abstract

Diverse chemical methods and comparative sequence analyses have identified Lys-166, Lys-329, and Glu-48 of Rubisco as active-site residues and have suggested that these residues participate in catalysis. These conclusions have been validated by site-directed mutagenesis; furthermore, hybridization of distinct site-directed mutant proteins demonstrates an intersubunit location of the active site in which side chains from each subunit are required. The ability of mutant proteins, devoid of overall carboxylase activity, to catalyze certain partial reactions suggests that Lys-166 is the base that enolizes ribulose-P/sub 2/ and that Lys-329 facilitates addition of gaseous substrate to the enediol. 39 refs., 6 figs., 2 tabs.

Authors:: Hartman, F C

Publication Date:: 1989-01-01

Research Org.:: Oak Ridge National Lab., TN (USA)

OSTI Identifier:: 5999278

Report Number(s):: CONF-8906166-1
ON: DE89014066

DOE Contract Number:: AC05-84OR21400

Resource Type:: Conference

Resource Relation:: Conference: NATO Advanced Study Institute summer school of enzymatic and model carboxylation and reduction reactions for carbon dioxide utilization, Bari, Italy, 17-28 Jun 1989; Other Information: Portions of this document are illegible in microfiche products

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; MOLECULAR STRUCTURE; DETECTION; PROTEINS; AMINO ACID SEQUENCE; BIOCHEMICAL REACTION KINETICS; CATALYSIS; HYBRIDIZATION; MUTAGENS; PROTEIN STRUCTURE; KINETICS; ORGANIC COMPOUNDS; REACTION KINETICS; 550200* - Biochemistry

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                    Hartman, F C. Rubisco: Active-site characterization and mechanistic implications.  United States: N. p., 1989. 
        Web.

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                    Hartman, F C. Rubisco: Active-site characterization and mechanistic implications.  United States.

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                    Hartman, F C. Sun .  
        "Rubisco: Active-site characterization and mechanistic implications".  United States.

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@article{osti_5999278,

  title        = {Rubisco: Active-site characterization and mechanistic implications},

  author       = {Hartman, F C},

  abstractNote = {Diverse chemical methods and comparative sequence analyses have identified Lys-166, Lys-329, and Glu-48 of Rubisco as active-site residues and have suggested that these residues participate in catalysis. These conclusions have been validated by site-directed mutagenesis; furthermore, hybridization of distinct site-directed mutant proteins demonstrates an intersubunit location of the active site in which side chains from each subunit are required. The ability of mutant proteins, devoid of overall carboxylase activity, to catalyze certain partial reactions suggests that Lys-166 is the base that enolizes ribulose-P/sub 2/ and that Lys-329 facilitates addition of gaseous substrate to the enediol. 39 refs., 6 figs., 2 tabs.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/5999278},
  journal      = {},
number       = ,

  volume       = ,

  place        = {United States},

  year         = {1989},

  month        = {1}

}

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Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction

Journal Article Hartman, F C ; Lee, E H - J. Biol. Chem.; (United States)

Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolizationmore »« less
Function of active-site residues of ribulose-bisphosphate carboxylase/oxygenase (Rubisco)

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Chemical modification, site-directed mutagenesis, and x-ray crystallography have identified Lys166, Lys329, and Glu48 as active-site residues of the homodimeric Rubisco from Rhodospirillum rubrum. (In the L/sub 8/S/sub 8/ Rubisco from spinach, these residues correspond to Lys175, Lys334, and Glu60.) We have used two strategies to explore the function of these residues: (a) introduction of very subtle structural changes into their side chains by combining chemical modification with site-directed mutagenesis and (b) evaluation of the ability of mutant proteins, devoid in overall carboxylase activity, to catalyze the enolization of D-ribulose-1,5-bisphosphate (RuBP), i.e. the first step in the normal reaction pathway. 15more » refs., 2 figs.« less
Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis

Conference Lee, E H ; Soper, T S ; Hartman, F C - Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50-kDa subunits. Although a high-resolution 3D structure of the enzyme has not yet been reported, recent work from their laboratory with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrated an intrasubunit distance of 12 A between the active-site lysines at positions 166 and 329. To continue mapping distances between residues in the active-site region, they have modified the enzyme with 4,4'-difluoro-3,3'-dinitrophenyl sulfone, which spans 9 A. The inactivated carboxylase exhibits an unaltered molecular weight as judged by gel filtration, thereby excluding intermolecular cross-linking. However, in the presence of urea, gel filtration reveals a prominent speciesmore »« less
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To test experimentally, the prior theoretical deduction that active-site residue Lys166 of ribulose-bisphosphate carboxylase participates in the carboxylation step of overall catalysis, site-directed mutants and chemically rescued site-directed mutants were characterized by kinetics and product analysis. Although position-166 mutants are able to catalyze normal enolization of ribulose bisphosphate, the enediol intermediate does not undergo carboxylation but rather eliminates phosphate. Furthermore, the chemically rescued mutant (aminoethylation of the severely impaired Lys66Cys mutant) generates a highly active mimic, which displays an enhanced carboxylation/oxygenation partition ratio. These two distinct lines of experimentation document a crucial role of Lys166 in carboxylation and in discriminationmore »« less
https://doi.org/10.2172/824531

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Partial reactions and chemical rescue of site-directed mutants of Rubisco as mechanistic probes

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Given the current state of knowledge of the reaction pathways catalyzed by D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the elucidation of the three-dimensional structure of several different forms of the enzyme, sit-directed mutagenesis offers the potential to decipher catalytic roles of active-site residues and to unravel the functional significance of various structural elements. Especially intriguing are intersubunit, electrostatic interactions at the active site between Glu48 and Lys168 of the nonactivated (noncarbamylated) enzyme and between Glu48 and Lys329 of the activated (carbamylated) enzyme. In this paper, we describe two approaches to address the roles of electrostatic interactions at the active site and themore »« less

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